Understanding the precise mechanistic role of phospholipase A2 in physiologically important regulatory processes is one of the most timely problems in biochemistry today. Although the absolute requirements for the enzyme in platelet aggregation, cardiac contraction and excitation, prostaglandin biosynthesis, as well as aldosterone dependent sodium transport has been definitively established, its precise in vivo function in these processes remains to be delineated. In order to provide the answers to these questions as well as to elucidate then enzyme's mechanism of action there is a vital need for highly specific and potent phospholipase inhibitors. Development of such inhibitors is the main objective of this research project. To realize our goal we intend to introduce a multifunctional approach: the inhibitors synthesized will be directed at more than one residue at the active site. They will include a number of anchoring groups to enhance specificity and binding; and one covalently reactive function per inhibitor to modify a single specific target residue at the active site. By varying the distance and orientation of the functional groups we intend to maximize the interaction between the enzyme and the inhibitor. The most potent sub-site directed inhibitors will be utilized for mapping the active site of the enzyme. As the approach is a general one once proven to work for phospholipase, it may be extendable to other enzymes as well. The advantage of this method is that unlike x-ray crystallography here we are mapping the active site in a dynamic state, therefore the structural information obtained is likely to approximate the position and the orientation of the active-site residues as they are involved in the catalytic process itself. Kinetic studies for determination of the efficiency of the inhibitors will be conducted using a bile salt-lecithin mixed-micelle assay-system. For further in vitro and in vivo testing of the inhibitors collaborative arrangements have been made. Nonenzymic model studies focusing on the mechanistic elucidation of dipolar aprotic acyl transfer reactions to "desolvated" carboxylate will be conducted to provide chemical precedents crucial for understanding the mechanism of the lipolytic reaction catalyzed by the enzyme.